An brief overview of UPLC including theory,stationary phases,instrumentation,aplications

1. (Ultra Pressure Liquid
Chromatography)
Presented by: Guided by:
Ms. Sonali R.Phadke Dr. Asha B. Thomas
M.Pharm :sem-I(QAT)
UPLC-An overview 1

2. Comparison between HPLC and
UPLC :
HPLC
 dp(3-10)µm
 Inlet pressure 5000 psi
 Lower precision comparative
in sample introduction.
 Detector that uses large flow
rates and larger detection
cells.
 Some are equipped with
automated sampling devices.
 Slower with lower resolution.
UPLC
 dp.(1.75-1.8)µm
 Inlet pressure more than
15000psi
 Higher precision in sample
introduction.
 Detector that uses small flow
rates and low detection
limits.
 Most of are equipped with
automated sampling devices.
 Rapid with higher resolution.
UPLC-An overview 2

3. The fundamental resolution
equation :
Rs=
𝑁
4
(
𝛼−1
𝛼
) (
𝑘
𝑘+1
)
efficiency selectivity retensivity
In UPLC, increasing N (efficiency) is the primary
focus.
As Rs is proportional to square root of N
Rs∝ √𝑁
If N ↑ 3𝑋 , 𝑅𝑠 ↑ 1.7𝑋
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4. Improving particle size:
Efficiency is inversely proportional to particle size so
N∝
1
𝑑𝑃
Rs ↑ 1.7X
(i.e. Rs∝ √𝑁)
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5. Resolution (constant column length):
Optimal flow rate is inversely proportional to dp
Fopt ∝ 1/𝑑𝑝
Isocratic analysis time is Inversely proportional to F
Therefore
T ↓3X
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6. Peak width and sensitivity
Efficiency E, is inversely proportional to square of peak
width.
N∝ 1/W2
Peak height is inversely proportional to peak width
Height ∝ 1/w
sensitivity ↑ 1.7 x
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7. Sensitivity increases:
 Assuming same efficiency, peak height is inversely
proportional to column length,
Height=1/L
For same length column length L decreased
proportional to particle size dp
(N=1x, Rs=1x)
Sensitivity ↑ 3𝑥
UPLC-An overview 7

8. Backpressure increases:
Backpressure (p) is directly proportional to column
length
P∝ 𝐿
For constant L/dp, Backpressure is inversely
proportional to the square to particle size,dp
P∝ 1/dp2
P↑ 𝟗𝑿
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9. HPLC vs UPLC
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10. Why to migrate from HPLC to UPLC?
 Acquire results in less time and
with more resolution.
 More information-faster
 More robust method-more
confidence
 Better situational response
 More samples analyzed per
system, per scientist
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11. UPLC stationary phases :
 Mechanical strength and stability of columns were
critical issues due to high pressure.
 Classical gel-sol method used for generation of
durable columns which are superior than silica
particles
 Made by incorporating carbon in form of methyl
groups
 Mechanically strong, better efficiency and can be used
over extended range of pH.
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12. Ethylene Hybrid Bridge(EHB) particles :
 This technology increases the stability of the 1.7 µm
particles by bridging the methyl group in the silica
matrix.
 Can withstand high pressure and pH.
 Efficiency of column is directly proportional to column
length and inversely proportional to particle size.
 Pore diameters are 130 Å and 300Å.
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13. High Strength Silica (HSS) particles
 Only UPLC certified
100% silica particles
having 1.8 𝜇
 These are tested for
15000 psi pressure
 Include tribonded C18
ligand
 Long column life and
UPLC efficiencies
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14. Column chemistries:
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15. Stationary phases:
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16. Charged surface Hybrid(CHB) technology
These particles
incorporate a low level
surface hybrid charge
improves sample
loadability and peak
asymmetry in low ionic
strength mobile phases.
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17. Instrumentation :
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18. • Acquity UPLC system consists :
 Binary solvent manager
Two individual serial flow pumps to deliver a parallel
binary gradient. provides steady pulse free solvent flow
at analytical flow rate(1-2 ml/min).
 Sample manager including sample heater
Injects sample draws from vial in to chromatographic
flow stream. Can perform injection approx. at 15 sec.
Also controls column temperature.
 Optional sample manager
Stores micro meter or vial plates and to and from the
sample manages, automating their processing and
increasing through put.
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19. Injecting the sample:
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20. Pumps: pumps
Constant
flow pumps
Reciprocating
piston pumps
Dual piston
pumps
Constant
pressure
pumps
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21. UPLC columns:
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22. Detectors:
detectors
Optical
detector
Tunable UV
detector
Evaporating
light scattering
detector
Florescence
detector
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23. Tunable UV detector:
 These are used for UV/visible
range
 Consists light guided flow
cell like optical fiber
 contains volume of 500
nanolitre and pathlength of
10 mm, and high sensitivity
flow cell with volume 2.4
microliters and 25 mm
pathlength.
 Operates at wavelength
ranging 190-700 nm
 Empower and MassLynx
software is used for
operations
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24. Applications:
 Identification of metabolites in in vitro discovery
studies.
 Bioequivalence study
 Detection of impurities
 Dissolution testing in relatively high potent drug
sample
 Method development/validation
 Forced degradation studies
 Impurity profiling
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25. References :
 Waters AAPS 2007 seminars, November 12-13,2007
 UPLC: a preeminent technique in pharmaceutical
analysis by Ashok Kumar et al, Acta Poloniae
Pharmaceutica-Drug research, vol.69 No.3pp.371-
380,2012
 Ultra Performance Liquid Chromatography: An
Indroduction And Review by T. Sunil Kumar Reddy et
al,IJPRA,vol 2,issue 1, 2012,24-31
 Ultra Performance Liquid Chromatography : A Review
by Patil V.P. et al.IRJP2(6) 2011 39-44
 Ultra Performance Liquid Chromatography: A
chromatography technique by Uttam Singh Baghel et
al.IJPQA.2010;2(1):19-25
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26. Thank you
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